Glutamate transporters (EAATs) play an important role in the regulation of extracellular levels of glutamate, a major excitatory neurotransmitter in the central nervous system. Current data suggest that increased extracellular glutamate concentration is involved in the pathophysiology of ischemic brain injury and several major human neurodegenerative disorders, such as Alzheimer's diseases and amyotrophic lateral sclerosis. Thus, normal functioning of EAATs is important for maintaining efficient neurotransmission and preventing neuronal injury. Our long-term research goals are to understand the regulation of EAATs and their role in the mechanisms of anesthesia, synaptic plasticity and neuroprotection. Volatile anesthetics, such as isoflurane, increase the activity and cell surface expression of EAAT type 3 (EAAT3), the major neuronal EAAT. This effect is protein kinase C (PKC) 1-dependent and relies on the phosphorylation of S465 in EAAT3. Isoflurane also increases the protein expression of EAAT3. This proposal aims to 1) determine whether isoflurane-induced EAAT3 redistribution to the plasma membrane is mediated by increased delivery of EAAT3 from cytosol to the plasma membrane;2) identify proteins involved in the recycling of EAAT3 and determine how phosphorylation of S465 changes the protein-protein interaction to alter the distribution of EAAT3;3) determine the role of S465 phosphorylation-induced EAAT3 redistribution in the formation of long-term potentiation and isoflurane-induced neuroprotection;and 4) identify the mechanisms for isoflurane to increase the EAAT3 expression. Cell cultures, hippocampal slices and intact animals (rats and mice) will be used in the studies. Protein biotinylation and immunoprecipitation will be performed to study EAAT3 trafficking and protein- protein interaction. RNA interference and dominant negative constructs will be used to down-regulate protein expression and functions. Sequence-specific peptide inhibitors will be used to investigate the biological functions of EAAT3 redistribution to the plasma membrane. EAAT3 promoter activity will be assayed in the presence or absence of isoflurane. These studies will provide a molecular basis for the anesthetic regulation of glutamate transport and may suggest new targets for neuroprotection or for regulation of synaptic plasticity. PUBLIC HEALTH RELEVANCE: Isoflurane, a commonly used anesthetic in clinical practice, affects the function and location of glutamate transporters, cellular proteins involved in a broad range of brain functions. This project is designed to investigate how isoflurane regulates the function and location of these proteins and the biological consequences of these regulations, such as providing brain adaptation and protection. These studies may help understand how isoflurane works to cause its biological effects, such as brain protection, and also may suggest novel methods to induce these effects.